RNA ligases are used for 3′-labeling of RNAs (the acceptor) by phosphorylated nucleotide analogs or oligonucleotides (the donor) in the presence of ATP (Aravin, 2005; Pfeffer, 2004). The reaction generally requires ATP because the donor molecule 5′ phosphate (p) needs to be adenylated by the RNA ligase. The RNA ligase subsequently positions the acceptor molecule 3′ hydroxyl terminus for attack on the adenylated donor phosphate (App) resulting in departure of the adenylate in the form of adenosine phosphate (AMP). The result is the formation of a 3′ acceptor/5′ donor phosphodiester linkage.
The requirement for ATP in ligation is eliminated if pre-adenylated compounds are provided (England, 1977). Non-nucleotidic pre-adenylated compounds can also be used as donor molecule substrates. Biotin or fluorescent dyes have been ligated to the 3′ end of tRNAs in this manner.
Most of the literature and commercial products use conventional T4 RNA ligase 1 (Rnl1), but more recently a second ligase has been described and characterized from phage T4, known as T4 RNA ligase 2 (Rnl2) (Ho and Shuman 2002). T4 Rnl2 is a 334 amino acid residue ligase that, like Rnl1, catalyzes intramolecular and intermolecular RNA strand ligation. In contrast to Rnl1, Rnl2 shows nick-sealing activity in a double-stranded RNA or an RNA-DNA context (Nandakumar et al. 2004). A truncated form of this ligase comprising amino acids 1-249 has been shown to maintain adenylyltransferase and AppRNA ligase activity. Deletion of amino acids 34 or 227 in full-length Rnl2 can inactivate the enzyme (Yin et al., 2003), indicating that N-terminal or C-terminal deletions of the enzyme beyond these points very likely would abolish ligase activity. Conservative mutation of residue K227 to Q rescues the activity of ligating pre-adenylated donor RNAs to acceptor RNAs but compromises the enzymes adenylate transfer activity. Some other residues, such as D120, K209, and K225 when mutated also differentially affect ligation of the pre-adenylated donor versus adenylate transfer activity (Yin et al., 2003).
Rnl2(1-249) has, due to its missing C-terminal domain, a reduced affinity for binding phosphate donors and therefore transfers the adenylate residue from the adenylated enzyme to the 5′-phosphate group of miRNA acceptors less efficiently then other ligases. Therefore, Rnl2(1-249) allows consistently better labeling results than Rnl1. Nevertheless, the ratio of desired ligation versus unwanted side reactions, such as circularization and dimerization, still depends on the kinetic parameters of individual steps of the ligase mechanism.
Circularization is a consequence of deadenylation of pre-adenylated donors followed by adenylate transfer to miRNA 5′ phosphates forming App-miRNA that will then circularize by attack of the miRNA 3′ hydroxyl and also dimerize to a certain degree.
Circularization can be partially suppressed by the use of high concentration donors or reduction of temperature but in cannot be avoided completely. These side reactions are mostly unpredictable and caused by sequence-dependent secondary structure variation of donor and acceptor molecules.
miRNAs are 21- to 23-nt RNA molecules that act as natural regulators of gene expression in plants and animals. In humans about 400 miRNA genes have been identified, and methods to characterize their tissue or cell-type specific expression patterns and their deregulation in disease are needed (Aravin, 2005). miRNAs are naturally 5′ phosphorylated and carry 2′,3′ dihydroxyl termini.
One of the approaches for detecting miRNAs is based on microarray hybridization that requires fluorescent labeling of the miRNA sample. An RNA ligase is used to conjugate a fluorescently labeled donor to the miRNA. However, the current methods of ligation are plagued by the unwanted side reactions described above.
Accordingly, a need exists for an improved RNA ligase enzyme that can more efficiently modify the 3′ position of RNA.